Gp91(phox) contributes to the development of experimental inflammatory bowel disease

Inflammatory bowel disease (IBD) is related to dysfunction of intestinal immunity. Neutrophils have an important role in innate immunity via the oxidative burst, using the p47phox- and gp91(phox)-containing NAD(P)H oxidase known as Nox2. In dextran sulphate sodium (DSS)-induced colitis, no significant difference in inflammation between p47(phox-/-) and wild-type (WT) mice was reported, but there was improved endothelium-dependent arteriolar dilation in gp91(phox-/-) mice, compared with that in WT mice. Gp91(phox) and p47 (phox) are not only essential components of phagocyte Nox2, but also have roles in other enzymes. Thus the differences in response of their respective gene knockout mice to DSS challenge are not completely unexpected, but need further investigation. The clinicopathological changes and immunological responses to DSS challenge have not been fully described in gp91(phox-/-) mice. Thus we treated WT and gp91(phox-/-) mice with 2.5% DSS for 7 days. The gp91(phox-/-) mice developed less severe colitis than WT mice following DSS treatment, reflected by a smaller body weight loss, less rectal bleeding and fewer histopathological changes. Less colonic myeloperoxidase was observed in gp91(phox-/-), compared with WT mice, following DSS challenge, correlating with interleukin (IL)-6 production. IL-10 was upregulated in both gp91(phox-/-) and WT mice, but was significantly higher in the latter, following 7 days DSS challenge. These results suggest that gp91(phox-/-) mice are less susceptible to acute DSS-induced colitis, possibly because of a reduced oxidative burst in the intestine and, consequently, less tissue damage.     

P-selectin-mediated adhesion impairs endothelium-dependent arteriolar dilation in hypercholesterolemic mice

Hypercholesterolemia is associated with an attenuation of endothelium-dependent dilation in arterioles and an increase in leukocyte and platelet adhesion in venules. The proximity of closely paired arterioles and venules is thought to facilitate heat and mass transport between the two and could be involved in transport of inflammatory and/or vasoactive mediators from venule to arteriole. In the current study, we tested the hypothesis that the impaired arteriolar dilation associated with hypercholesterolemia might be dependent on P-selectin-dependent blood cell adhesion in the closely paired venules. Leukocyte and platelet recruitment in venules and the endothelium-dependent response to bradykinin in second-order arterioles were observed in the mouse intestinal submucosa using intravital microscopy. Four weeks of a high-cholesterol diet decreased bradykinin-induced arteriolar dilation more dramatically in closely paired arterioles than in distantly paired arterioles. The dysfunctional arteriolar dilation of closely paired arterioles in hypercholesterolemic mice was significantly improved when the experiments were repeated in P-selectin-deficient mice (given the high-cholesterol diet) or in hypercholesterolemic mice injected with a P-selectin monoclonal antibody. A similar improvement in dilation of closely paired arterioles was attained in hypercholesterolemic mice given the superoxide dismutase mimetic Tempol. These findings indicate that hypercholesterolemia-induced increases in venular leukocyte and platelet adhesion might contribute to the impaired endothelium-dependent dilation of closely paired arterioles via a mechanism that is distance limited and dependent on P-selectin and superoxide.     

Chronic exposure to nicotine alters endothelium-dependent arteriolar dilatation: effect of superoxide dismutase.

The first goal of this study was to determine whether chronic injection of nicotine alters endothelium-dependent arteriolar dilatation. We measured the diameter of cheek pouch resistance arterioles (approximately 50 microm in diameter) in response to endothelium-dependent (acetylcholine and ADP) and -independent (nitroglycerin) agonists in control hamsters and hamsters treated with nicotine (2 microg. kg-1. day-1 for 2-3 wk). In control hamsters, acetylcholine (0.1 and 1.0 microM) dilated arterioles by 13 +/- 2 and 31 +/- 3%, respectively, and ADP (1.0 and 10 microM) dilated arterioles by 18 +/- 1 and 30 +/- 1%, respectively. In contrast, acetylcholine (0.1 and 1.0 microM) dilated arterioles by only 5 +/- 2 and 12 +/- 3%, respectively, and ADP (1.0 and 10 microM) dilated arterioles by only 7 +/- 2 and 13 +/- 3%, respectively, in animals treated with nicotine (P < 0.05 vs. response in control hamsters). Nitroglycerin produced similar dose-related dilatation of cheek pouch arterioles in control and nicotine-treated hamsters. Our second goal was to examine a possible mechanism for impaired endothelium-dependent arteriolar dilatation during chronic treatment with nicotine. We found that superfusion of the cheek pouch microcirculation with superoxide dismutase (150 U/ml) restored impaired endothelium-dependent, but did not alter endothelium-independent, arteriolar dilatation in hamsters treated with nicotine. Superfusion with superoxide dismutase did not alter endothelium-dependent or -independent arteriolar dilatation in control hamsters. We suggest that chronic exposure to nicotine produces selective impairment of endothelium-dependent arteriolar dilatation via a mechanism related to the synthesis/release of oxygen-derived free radicals.     

Mechanisms of platelet and leukocyte recruitment in experimental colitis

Both leukocytes and platelets accumulate in the colonic microvasculature during experimental colitis, leading to microvascular dysfunction and tissue injury. The objective of this study was to determine whether the recruitment of leukocytes and platelets in inflamed colonic venules are codependent processes. The rolling and adherence of leukocytes and platelets in colonic venules of mice with dextran sodium sulfate (DSS)-induced colitis were monitored by intravital videomicroscopy. DSS elicited an increased recruitment of both rolling and adherent leukocytes and platelets. DSS-colitic mice rendered thrombocytopenic with anti-platelet serum exhibited profound reductions in leukocyte adhesion. Neutropenia, induced with anti-neutrophil serum, significantly reduced the adhesion of leukocytes and the accumulation of platelet-leukocyte aggregates while greatly enhancing the number of platelets that roll and adhere directly to venular endothelial cells. The enhanced platelet adhesion associated with neutropenia was mediated by platelet P-selectin interactions with endothelial cell P-selectin glycoprotein ligand (PSGL-1). DSS colitis was also associated with an increased expression of PSGL-1 in the colonic vasculature. These findings indicate that the recruitment of leukocytes and platelets in inflamed colonic venules are co-dependent processes.     

Venular-arteriolar communication in the regulation of blood flow

Muscle blood flow is regulated to meet the metabolic needs of the tissue. With the vasculature arranged as a successive branching of arterioles and the larger, >50 microm, arterioles providing the major site of resistance, an increasing metabolic demand requires the vasodilation of the small arterioles first then the vasodilation of the more proximal, larger arterioles. The mechanism(s) for the coordination of this ascending vasodilation are not clear and may involve a conducted vasodilation and/or a flow-dependent response. The close arteriolar-venular pairing provides an additional mechanism by which the arteriolar diameter can be increased due to the diffusion of vasoactive substances from the venous blood. Evidence is presented that the venular endothelium releases a relaxing factor, a metabolite of arachidonic acid, that will vasodilate the adjacent arteriole. The stimulus for this release is not known, but it is hypothesized that hypoxia-induced ATP release from red blood cells may be responsible for the stimulation of arachidonic release from the venular endothelial cells. Thus the venous circulation is in an optimal position to monitor the overall metabolic state of the tissue and thus provide a feedback regulation of arteriolar diameter.     

Osteopontin as two-sided mediator of intestinal inflammation

Osteopontin (OPN) is characterized as a major amplifier of Th1-immune responses. However, its role in intestinal inflammation is currently unknown. We found considerably raised OPN levels in blood of wild-type (WT) mice with dextran sodium sulfate (DSS)-induced colitis. To identify the role of this mediator in intestinal inflammation, we analysed experimental colitis in OPN-deficient (OPN(-/-)) mice. In the acute phase of colitis these mice showed more extensive colonic ulcerations and mucosal destruction than WT mice, which was abrogated by application of soluble OPN. Within the OPN(-/-) mice, infiltrating macrophages were not activated and showed impaired phagocytosis. Reduced mRNA expression of interleukin (IL)-1 beta and matrix metalloproteinases was found in acute colitis of OPN(-/-) mice. This was associated with decreased blood levels of IL-22, a Th17 cytokine that may mediate epithelial regeneration. However, OPN-(/-) mice showed increased serum levels of tumour necrosis factor (TNF)-alpha, which could be due to systemically present lipopolysaccharide translocated to the gut. In contrast to acute colitis, during chronic DSS-colitis, which is driven by a Th1 response of the lamina propria infiltrates, OPN(-/-) mice were protected from mucosal inflammation and demonstrated lower serum levels of IL-12 than WT mice. Furthermore, neutralization of OPN in WT mice abrogated colitis. Lastly, we demonstrate that in patients with active Crohn's disease OPN serum concentration correlated significantly with disease activity. Taken together, we postulate a dual function of OPN in intestinal inflammation: During acute inflammation OPN seems to activate innate immunity, reduces tissue damage and initiates mucosal repair whereas during chronic inflammation it promotes the Th1 response and strengthens inflammation.     

Platelet-associated NAD(P)H oxidase contributes to the thrombogenic phenotype induced by hypercholesterolemia

Elevated cholesterol levels promote proinflammatory and prothrombogenic responses in venules and impaired endothelium-dependent arteriolar dilation. Although NAD(P)H oxidase-derived superoxide has been implicated in the altered vascular responses to hypercholesterolemia, it remains unclear whether this oxidative pathway mediates the associated arteriolar dysfunction and platelet adhesion in venules. Platelet and leukocyte adhesion in cremasteric postcapillary venules and arteriolar dilation responses to acetylcholine were monitored in wild-type (WT), Cu,Zn-superoxide dismutase transgenic (SOD-TgN), and NAD(P)H oxidase-knockout (gp91(phox-/-)) mice placed on a normal (ND) or high-cholesterol (HC) diet for 2 weeks. HC elicited increased platelet and leukocyte adhesion in WT mice versus ND. Cytosolic subunits of NAD(P)H oxidase (p47phox and p67phox) were expressed in platelets. This was not altered by hypercholesterolemia; however, platelets and leukocytes from HC mice exhibited elevated generation of reactive oxygen species compared to ND mice. Hypercholesterolemia-induced leukocyte recruitment was attenuated in SOD-TgN-HC and gp91(phox-/-)-HC mice. Recruitment of platelets derived from WT-HC mice in venules of SOD-TgN-HC or gp91(phox-/-)-HC recipients was comparable to ND levels. Adhesion of SOD-TgN-HC platelets paralleled the leukocyte response and was attenuated in SOD-TgN-HC recipients, but not in WT-HC recipients. However, gp91(phox-/-)-HC platelets exhibited low levels of adhesion comparable to those of WT-ND in both hypercholesterolemic gp91(phox-/-) and WT recipients. Arteriolar dysfunction was evident in WT-HC mice, compared to WT-ND. Overexpression of SOD or, to a lesser extent, gp91(phox) deficiency restored arteriolar vasorelaxation responses toward WT-ND levels. These findings reveal a novel role for platelet-associated NAD(P)H oxidase in producing the thrombogenic phenotype in hypercholesterolemia and demonstrate that NAD(P)H oxidase-derived superoxide mediates the HC-induced arteriolar dysfunction.     

NAD(P)H oxidase and eNOS play differential roles in cytomegalovirus infection-induced microvascular dysfunction

Primary cytomegalovirus (CMV) infection promotes oxidative stress and reduces nitric oxide (NO) bioavailability in endothelial cells. These events are among the earliest vascular responses to cardiovascular risk factors. We assessed the roles of NAD(P)H oxidase and NO bioavailability in microvascular responses to persistent CMV infection alone or with hypercholesterolemia. Wild-type (WT) or gp91^phox (NAD(P)H oxidase subunit) knockout mice received mock inoculum or 3 × 10⁴ PFU murine CMV (mCMV) ip 5 weeks before placement on a normal or high-cholesterol diet (HC) for 4 weeks before assessment of arteriolar function and venular blood cell recruitment using intravital microscopy. Some WT groups received sepiapterin (a precursor of the nitric oxide synthase cofactor tetrahydrobiopterin) or apocynin (NAD(P)H oxidase inhibitor/antioxidant). Endothelium-dependent vasodilation was impaired in mCMV vs mock WT, regardless of diet. This was not affected by sepiapterin, and pharmacological inhibition of nitric oxide synthase reduced dilation similarly in mock and mCMV mice. Apocynin or deficiency of total, but not blood cell or vascular wall only (tested using bone marrow chimeras), gp91^phox protected against arteriolar dysfunction. Blood cell recruitment was induced by mCMV–HC. Sepiapterin, but not NAD(P)H oxidase deficiency/apocynin, reduced leukocyte accumulation, whereas platelet adhesion was reduced by sepiapterin, apocynin, or total, platelet-specific, or vascular wall gp91^phox deficiency. These data implicate activation of both hematopoietic and vessel wall NAD(P)H oxidase in mCMV-induced arteriolar dysfunction and platelet and vascular NAD(P)H oxidase in the thrombogenic phenotype induced by mCMV–HC. In contrast, findings with sepiapterin suggest that eNOS dysfunction, perhaps uncoupling, mediates venular, but not arteriolar, responses to mCMV–HC, thus indicating that NAD(P)H oxidase and eNOS differentially regulate microvascular responses to mCMV.     

Milk fat globule-EGF factor 8 is a critical protein for healing of dextran sodium sulfate-induced acute colitis in mice

Milk fat globule-EGF factor 8 (MFG-E8) has been shown to play an important role in maintaining the integrity of the intestinal mucosa and to accelerate healing of the mucosa in septic mice. Herein, we (a) analyzed the expression of MFG-E8 in the gut of wild-type (WT) C57BL/6 (MFG-E8(+/+)) mice with and without dextran sulfate sodium (DSS)-induced colitis, (b) characterized the pathological changes in intestinal mucosa of MFG-E8(+/+) and MFG-E8(-/-) mice with DSS-induced colitis and (c) examined the therapeutic role of MFG-E8 in inflammatory bowel disease by using DSS-induced colitis model. Our data documented that there was an increase in colonic and rectal MFG-E8 expression in MFG-E8(+/+) mice during the development of DSS colitis. MFG-E8 levels in both tissues decreased to below baseline during the recovery phase in mice with colitis. Changes in MFG-E8 gene expression correlated to the levels of inflammatory response and crypt-epithelial injury in both colonic and rectal mucosa in MFG-E8(+/+) mice. MFG-E8(-/-)mice developed more severe crypt-epithelial injury than MFG-E8(+/+) mice during exposure to DSS with delayed healing of intestinal epithelium during the recovery phase of DSS colitis. Administration of MFG-E8 during the recovery phase ameliorated colitis and promoted mucosal repair in both MFG-E8(-/-) and MFG-E8(+/+) mice, indicating that lack of MFG-E8 causes increased susceptibility to colitis and delayed mucosal healing. These data suggest that MGF-E8 is an essential protective factor for gut epithelial homeostasis, and exogenous administration of MFG-E8 may represent a novel therapeutic target in inflammatory bowel disease.     

oxLDL specifically impairs endothelium-dependent, NO-mediated dilation of coronary arterioles

Our previous studies implicated that oxidized low-density lipoprotein (oxLDL), a putative atherogenic agent, impairs endothelium-dependent, nitric oxide (NO)-mediated dilation of isolated coronary arterioles to pharmacological agonists. However, it is not known whether oxLDL specifically affects NO-mediated dilation or generally impairs endothelium-dependent function, including the release of hyperpolarizing factors. In this regard, we investigated the dilation of isolated porcine coronary arterioles (50- to 100-microm luminal diameter) in response to the activation of various endothelium-dependent pathways before and after intraluminal incubation of the vessels with oxLDL (0.5 mg protein/ml for 60 min). In the absence of oxLDL, all vessels developed basal tone and dilated in response to the activation of NO synthase (by flow and adenosine), cyclooxygenase (by arachidonic acid), cytochrome P-450 monooxygenase (by bradykinin), and endothelial membrane hyperpolarization (by sucrose-induced hyperosmolarity). Incubation of the vessels with oxLDL for 60 min did not alter basal tone but did inhibit the vasodilatory responses to increased flow and adenosine in a manner similar to that of the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester. Vasodilations in response to flow and adenosine were not affected by intraluminal incubation of the vessels with either a vehicle solution or the native LDL (0.5 mg protein/ml, 60 min). In contrast with the NO-mediated response, hyperosmotic vasodilation mediated by endothelial hyperpolarization was not affected by oxLDL. Endothelium-dependent dilations to the cyclooxygenase activator arachidonic acid and to the cytochrome P-450 monooxygenase activator bradykinin and endothelium-independent vasodilation to sodium nitroprusside were also not altered by oxLDL. Collectively, these results indicate that oxLDL has a selective effect on endothelium-dependent dilation with specific impairment of the NO-mediated response, whereas cyclooxygenase and cytochrome P-450 monooxygenase-mediated dilations are spared from this inhibitory effect. In addition, oxLDL does not appear to affect vasodilation mediated by hyperpolarization of the endothelium.     

Vasoactive Intestinal Polypeptide Promotes Intestinal Barrier Homeostasis and Protection Against Colitis in Mice

Inflammatory bowel disease is a chronic gastrointestinal inflammatory disorder associated with changes in neuropeptide expression and function, including vasoactive intestinal peptide (VIP). VIP regulates intestinal vasomotor and secretomotor function and motility; however, VIP’s role in development and maintenance of colonic epithelial barrier homeostasis is unclear. Using VIP deficient (VIPKO) mice, we investigated VIP’s role in epithelial barrier homeostasis, and susceptibility to colitis. Colonic crypt morphology and epithelial barrier homeostasis were assessed in wildtype (WT) and VIPKO mice, at baseline. Colitic responses were evaluated following dinitrobenzene sulfonic acid (DNBS) or dextran-sodium sulfate (DSS) exposure. Mice were also treated with exogenous VIP. At baseline, VIPKO mice exhibited distorted colonic crypts, defects in epithelial cell proliferation and migration, increased apoptosis, and altered permeability. VIPKO mice also displayed reduced goblet cell numbers, and reduced expression of secreted goblet cell factors mucin 2 and trefoil factor 3. These changes were associated with reduced expression of caudal type homeobox 2 (Cdx2), a master regulator of intestinal function and homeostasis. DNBS and DSS-induced colitis were more severe in VIPKO than WT mice. VIP treatment rescued the phenotype, protecting VIPKO mice against DSS colitis, with results comparable to WT mice. In conclusion, VIP plays a crucial role in the development and maintenance of colonic epithelial barrier integrity under physiological conditions and promotes epithelial repair and homeostasis during colitis.     

Diminished nitroprusside-induced relaxation of inflamed colonic smooth muscle in mice.

The dextran sodium sulphate (DSS) induced colitis in mice was used as a experimental model to study the contractility of murine longitudinal colonic smooth muscle during inflammation. Smooth muscle segments of proximal, middle and distal colon were mounted in organ baths. Smooth muscle contraction was induced by carbachol showing an aboral increase in activity, whereas in the inflamed middle colonic segment a marked decrease in activity was observed. The dilatative effect of sodium-nitroprusside (SNP) as a nitric oxide donor was investigated after precontraction by carbachol. Both in normal and DSS segments administration of SNP to isolated mouse colonic smooth muscle preparations caused regional differences in relaxation, the highest relaxation seen in normal proximal colonic tissue. However, this relaxation was markedly reduced in inflamed proximal preparations, associated with a diminished cGMP contents.     

EP4 Receptor–Associated Protein in Macrophages Ameliorates Colitis and Colitis-Associated Tumorigenesis

Prostaglandin E₂ plays important roles in the maintenance of colonic homeostasis. The recently identified prostaglandin E receptor (EP) 4–associated protein (EPRAP) is essential for an anti-inflammatory function of EP4 signaling in macrophages in vitro. To investigate the in vivo roles of EPRAP, we examined the effects of EPRAP on colitis and colitis-associated tumorigenesis. In mice, EPRAP deficiency exacerbated colitis induced by dextran sodium sulfate (DSS) treatment. Wild-type (WT) or EPRAP-deficient recipients transplanted with EPRAP-deficient bone marrow developed more severe DSS-induced colitis than WT or EPRAP-deficient recipients of WT bone marrow. In the context of colitis-associated tumorigenesis, both systemic EPRAP null mutation and EPRAP-deficiency in the bone marrow enhanced intestinal polyp formation induced by azoxymethane (AOM)/DSS treatment. Administration of an EP4-selective agonist, ONO-AE1-329, ameliorated DSS-induced colitis in WT, but not in EPRAP-deficient mice. EPRAP deficiency increased the levels of the phosphorylated forms of p105, MEK, and ERK, resulting in activation of stromal macrophages in DSS-induced colitis. Macrophages of DSS-treated EPRAP-deficient mice exhibited a marked increase in the expression of pro-inflammatory genes, relative to WT mice. By contrast, forced expression of EPRAP in macrophages ameliorated DSS-induced colitis and AOM/DSS-induced intestinal polyp formation. These data suggest that EPRAP in macrophages functions crucially in suppressing colonic inflammation. Consistently, EPRAP-positive macrophages were also accumulated in the colonic stroma of ulcerative colitis patients. Thus, EPRAP may be a potential therapeutic target for inflammatory bowel disease and associated intestinal tumorigenesis.     

Mechanisms underlying the anti-inflammatory actions of boswellic acid derivatives in experimental colitis

Recent clinical trials of the gum resin of Boswellia serrata have shown promising results in patients with ulcerative colitis. The objective of this study was to determine whether a semisynthetic form of acetyl-11-keto-beta-boswellic acid (sAKBA), the most potent anti-inflammatory component of the resin, also confers protection in experimental murine colitis induced by dextran sodium sulfate (DSS) to compare its effects with those standard medications of ulcerative colitis like steroids and to examine whether leukocyte-endothelial cell adhesion is a major target of action of sAKBA. Clinical measurements of disease activity and histology were used to assess disease progression, and intravital microscopy was employed to monitor the adhesion of leukocytes and platelets in postcapillary venules of the inflamed colon. sAKBA treatment significantly blunted disease activity as assessed both grossly and by histology. Similarly, the recruitment of adherent leukocytes and platelets into inflamed colonic venules was profoundly reduced in mice treated with sAKBA. Because previous studies in the DSS model have shown that P-selectin mediates these blood cell-endothelial cell interactions, the expression of P-selectin in the colonic microcirculation was monitored using the dual-radiolabeled antibody technique. The treatment of established colitis with sAKBA largely prevented the P-selectin upregulation normally associated with DSS colitis. All of the protective responses observed with sAKBA were comparable to that realized in mice treated with a corticosteroid. Our findings demonstrated an anti-inflammatory effect of sAKBA and indicated that P-selectin-mediated recruitment of inflammatory cells is a major site of action for this novel anti-inflammatory agent.     

Cellular glutathione peroxidase deficiency and endothelial dysfunction

Cellular glutathione peroxidase (GPx-1) is the most abundant intracellular isoform of the GPx antioxidant enzyme family. In this study, we hypothesized that GPx-1 deficiency directly induces an increase in vascular oxidant stress, with resulting endothelial dysfunction. We studied vascular function in a murine model of homozygous deficiency of GPx-1 (GPx-1(-/-)). Mesenteric arterioles of GPx-1(-/-) mice demonstrated paradoxical vasoconstriction to beta-methacholine and bradykinin, whereas wild-type (WT) mice showed dose-dependent vasodilation in response to both agonists. One week of treatment of GPx-1(-/-) mice with L-2-oxothiazolidine-4-carboxylic acid (OTC), which increases intracellular thiol pools, resulted in restoration of normal vascular reactivity in the mesenteric bed of GPx-1(-/-) mice. We observed an increase of the isoprostane iPF(2alpha)-III, a marker of oxidant stress, in the plasma and aortas of GPx-1(-/-) mice compared with WT mice, which returned toward normal after OTC treatment. Aortic sections from GPx-1(-/-) mice showed increased binding of an anti-3-nitrotyrosine antibody in the absence of frank vascular lesions. These findings demonstrate that homozygous deficiency of GPx-1 leads to impaired endothelium-dependent vasodilator function presumably due to a decrease in bioavailable nitric oxide and to increased vascular oxidant stress. These vascular abnormalities can be attenuated by increasing bioavailable intracellular thiol pools.     

Restoration of coronary endothelial function in obese Zucker rats by a low-carbohydrate diet

A popular diet used for weight reduction is the low-carbohydrate diet, which has most calories derived from fat and protein, but effects of this dietary regimen on coronary vascular function have not been identified. We tested the hypothesis that obesity-induced impairment in coronary endothelial function is reversed by a low-carbohydrate diet. We used four groups of male Zucker rats: lean and obese on normal and low-carbohydrate diets. Rats were fed ad libitum for 3 wk; total caloric intake and weight gain were similar in both diets. To assess endothelial and vascular function, coronary arterioles were cannulated and pressurized for diameter measurements during administration of acetylcholine or sodium nitroprusside or during flow. When compared with lean rats, endothelium-dependent acetylcholine-induced vasodilation was impaired by approximately 50% in obese rats (normal diet), but it was restored to normal by the low-carbohydrate diet. When the normal diet was fed, flow-induced dilation (FID) was impaired by >50% in obese compared with lean rats. Similar to acetylcholine, responses to FID were restored to normal by a low-carbohydrate diet. N(omega)-nitro-L-arginine methyl ester (10 microM), an inhibitor of nitric oxide (NO) synthase, inhibited acetylcholine- and flow-induced dilation in lean rats, but it had no effect on acetylcholine- or flow-induced vasodilation in obese rats on a low-carbohydrate diet. Tetraethylammonium, a nonspecific K(+) channel antagonist, blocked flow-dependent dilation in the obese rats, suggesting that the improvement in function was mediated by a hyperpolarizing factor independent of NO. In conclusion, obesity-induced impairment in endothelium-dependent vasodilation of coronary arterioles can be dramatically improved with a low-carbohydrate diet most likely through the production of a hyperpolarizing factor independent of NO.     

Functional contribution of P2Y1 receptors to the control of coronary blood flow

Activation of ADP-sensitive P2Y(1) receptors has been proposed as an integral step in the putative "nucleotide axis" regulating coronary blood flow. However, the specific mechanism(s) and overall contribution of P2Y(1) receptors to the control of coronary blood flow have not been clearly defined. Using vertically integrative studies in isolated coronary arterioles and open-chest anesthetized dogs, we examined the hypothesis that P2Y(1) receptors induce coronary vasodilation via an endothelium-dependent mechanism and contribute to coronary pressure-flow autoregulation and/or ischemic coronary vasodilation. Immunohistochemistry revealed P2Y(1) receptor expression in coronary arteriolar endothelial and vascular smooth muscle cells. The ADP analog 2-methylthio-ADP induced arteriolar dilation in vitro and in vivo that was abolished by the selective P2Y(1) antagonist MRS-2179 and the nitric oxide synthase inhibitor N(G)-nitro-l-arginine methyl ester. MRS-2179 did not alter baseline coronary flow in vivo but significantly attenuated coronary vasodilation to ATP in vitro and in vivo and the nonhydrolyzable ATP analog ATPγS in vitro. Coronary blood flow responses to alterations in coronary perfusion pressure (40-100 mmHg) or to a brief 15-s coronary artery occlusion were unaffected by MRS-2179. Our data reveal that P2Y(1) receptors are functionally expressed in the coronary circulation and that activation produces coronary vasodilation via an endothelium/nitric oxide-dependent mechanism. Although these receptors represent a critical component of purinergic coronary vasodilation, our findings indicate that P2Y(1) receptor activation is not required for coronary pressure-flow autoregulation or reactive hyperemia.     

GPR65 (TDAG8) inhibits intestinal inflammation and colitis-associated colorectal cancer development in experimental mouse models

GPR65 (TDAG8) is a proton-sensing G protein-coupled receptor predominantly expressed in immune cells. Genome-wide association studies (GWAS) have identified GPR65 gene polymorphisms as an emerging risk factor for the development of inflammatory bowel disease (IBD). Patients with IBD have an elevated risk of developing colorectal cancer when compared to the general population. To study the role of GPR65 in intestinal inflammation and colitis-associated colorectal cancer (CAC), colitis and CAC were induced in GPR65 knockout (KO) and wild-type (WT) mice using dextran sulfate sodium (DSS) and azoxymethane (AOM)/DSS, respectively. Disease severity parameters such as fecal score, colon shortening, histopathology, and mesenteric lymph node enlargement were aggravated in GPR65 KO mice compared to WT mice treated with DSS. Elevated leukocyte infiltration and fibrosis were observed in the inflamed colon of GPR65 KO when compared to WT mice which may represent a cellular mechanism for the observed exacerbation of intestinal inflammation. In line with high expression of GPR65 in infiltrated leukocytes, GPR65 gene expression was increased in inflamed intestinal tissue samples of IBD patients compared to normal intestinal tissues. Moreover, colitis-associated colorectal cancer development was higher in GPR65 KO mice than WT mice when treated with AOM/DSS. Altogether, our data demonstrate that GPR65 suppresses intestinal inflammation and colitis-associated tumor development in murine colitis and CAC models, suggesting potentiation of GPR65 with agonists may have an anti-inflammatory therapeutic effect in IBD and reduce the risk of developing colitis-associated colorectal cancer.     

Efficacy of an inhibitor of adhesion molecule expression (GI270384X) in the treatment of experimental colitis

Modulation of adhesion molecule expression or function is regarded as a promising therapy for inflammatory conditions. This study evaluates the effects of an inhibitor of adhesion molecule expression (GI270384X) in two experimental models of colitis. Colitis of different severity was induced in C57BL/6J mice by administering 1, 2, or 3% dextran sulfate sodium (DSS). GI270384X (3, 10, or 25 mg.kg(-1).day(-1)) was administered as pretreatment or started 3 days after colitis induction. In IL-10-deficient mice, the highest dose was given for 2 wk. The clinical course of colitis, pathological changes, serum inflammatory biomarkers, expression of adhesion molecules, and leukocyte-endothelial cell interactions in colonic venules were measured in mice treated with vehicle or with active drug. In the most severe forms of colitis (2% and 3% DSS and IL-10-deficient mice), the magnitude of colonic inflammation was not modified by treatment with GI270384X. In a less severe form of colitis (1% DSS), GI270384X treatment dose dependently ameliorated the clinical signs of colitis, colonic pathological changes, and serum levels of biomarkers (IL-6 and serum amyloid A). Administration of 25 mg.kg(-1).day(-1) GI270384X abrogated upregulation of ICAM-1 in the inflamed colon but had no effect on VCAM-1 or E-selectin expression. This was associated with a significant reduction in number of rolling and firmly adherent leukocytes in colonic venules. These results indicate that GI270384X is effective in the treatment of experimental colitis of moderate severity. Reduced adhesion molecule expression and leukocyte recruitment to the inflamed intestine contribute to this beneficial effect.     

Factor XIII Transglutaminase Supports the Resolution of Mucosal Damage in Experimental Colitis

The thrombin-activated transglutaminase factor XIII (FXIII) that covalently crosslinks and stablizes provisional fibrin matrices is also thought to support endothelial and epithelial barrier function and to control inflammatory processes. Here, gene-targeted mice lacking the FXIII catalytic A subunit were employed to directly test the hypothesis that FXIII limits colonic pathologies associated with experimental colitis. Wildtype (WT) and FXIII^-/- mice were found to be comparable in their initial development of mucosal damage following exposure to dextran sulfate sodium (DSS) challenge. However, unlike FXIII-sufficient mice, FXIII-deficient cohorts failed to efficiently resolve colonic inflammatory pathologies and mucosal damage following withdrawal of DSS. Consistent with prior evidence of ongoing coagulation factor activation and consumption in individuals with active colitis, plasma FXIII levels were markedly decreased in colitis-challenged WT mice. Treatment of colitis-challenged mice with recombinant human FXIII-A zymogen significantly mitigated weight loss, intestinal bleeding, and diarrhea, regardless of whether cohorts were FXIII-sufficient or were genetically devoid of FXIII. Similarly, both qualitative and quantitative microscopic analyses of colonic tissues revealed that exogenous FXIII improved the resolution of multiple colitis disease parameters in both FXIII^-/- and WT mice. The most striking differences were seen in the resolution of mucosal ulceration, the most severe histopathological manifestation of DSS-induced colitis. These findings directly demonstrate that FXIII is a significant determinant of mucosal healing and clinical outcome following inflammatory colitis induced mucosal injury and provide a proof-of-principle that clinical interventions supporting FXIII activity may be a means to limit colitis pathology and improve resolution of mucosal damage.