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Cyclooxygenase-2 (COX-2) is an inducible enzyme expressed in inflammation and cancer targeted by nonsteroidal anti-inflammatory drugs. COX-2 is also expressed constitutively in discreet locations where its inhibition drives gastrointestinal and cardiovascular/renal side effects. Constitutive COX-2 expression in the kidney regulates renal function and blood flow; however, the global relevance of the kidney versus other tissues to COX-2–dependent blood flow regulation is not known. Here, we used a microsphere deposition technique and pharmacological COX-2 inhibition to map the contribution of COX-2 to regional blood flow in mice and compared this to COX-2 expression patterns using luciferase reporter mice. Across all tissues studied, COX-2 inhibition altered blood flow predominantly in the kidney, with some effects also seen in the spleen, adipose, and testes. Of these sites, only the kidney displayed appreciable local COX-2 expression. As the main site where COX-2 regulates blood flow, we next analyzed the pathways involved in kidney vascular responses using a novel technique of video imaging small arteries in living tissue slices. We found that the protective effect of COX-2 on renal vascular function was associated with prostacyclin signaling through PPARβ/δ (peroxisome proliferator-activated receptor-β/δ). These data demonstrate the kidney as the principle site in the body where local COX-2 controls blood flow and identifies a previously unreported PPARβ/δ-mediated renal vasodilator pathway as the mechanism. These findings have direct relevance to the renal and cardiovascular side effects of drugs that inhibit COX-2, as well as the potential of the COX-2/prostacyclin/PPARβ/δ axis as a therapeutic target in renal disease. MOE (Min. of Education, S’pore) Published version

Alum-independent OVA-induced airway hyperresponsiveness to 5-HT and IgE production. Male C57Bl/6 mice were sensitised with saline or OVA in the presence or absence of Alum and subsequently challenged with saline or OVA. Animals sensitised in the absence of Alum were placed in WBP chambers and airway responsiveness to 5-HT was assessed 3 days after final challenge (C). Data (n = 7–9) was expressed as mean Penh AUC ± S.E.M. Plasma levels of total IgE (A) and OVA-specific IgE (B) were assessed by ELISA 3 days after final OVA challenge. Data (n = 7–9) expressed as mean ± S.E.M. *p

Ig levels in response to sensitisation with HDM via different routes. Male C57bl/6 mice were sensitised with vehicle or HDM (5 μg/kg HDM) either i.n. or i.p. (with or without Alum). Levels of HDM-specific IgE (A), total IgG1 (B) and HDM-specific IgG1 (C) in plasma were measured by ELISA. Data (n = 6) expressed as means ± S.E.M. *p

Alum-independent OVA-induced airway hyperresponsiveness to 5-HT and IgE production. Male C57Bl/6 mice were sensitised with saline or OVA in the presence or absence of Alum and subsequently challenged with saline or OVA. Animals sensitised in the absence of Alum were placed in WBP chambers and airway responsiveness to 5-HT was assessed 3 days after final challenge (C). Data (n = 7–9) was expressed as mean Penh AUC ± S.E.M. Plasma levels of total IgE (A) and OVA-specific IgE (B) were assessed by ELISA 3 days after final OVA challenge. Data (n = 7–9) expressed as mean ± S.E.M. *p

The effect of intranasal HDM challenge in topically sensitised mice. Male C57bl/6 mice were sensitised i.n. or i.p. as indicated below the figures with saline or HDM. Mice were subsequently challenged with saline or HDM. 3 days after the last challenge lungs were lavaged and BALF eosinophil (A), neutrophil (B) and lymphocyte (C) numbers were determined. Data (n = 8–12) expressed as mean cell number (103/ml) ± S.E.M. *p

Effect of HDM sensitisation and challenge on airway responsiveness to inhaled 5-HT. Male C57bl/6 mice were sensitised with saline or HDM (no Alum) and subsequently challenged with saline or HDM. Anaesthetised mice were instrumented and airway responsiveness to inhaled 5-HT was assessed by changes in resistance. Data (n = 6) expressed as mean resistance ± S.E.M. (DOC 150 kb)

Effect of HDM sensitisation and challenge on airway responsiveness to inhaled 5-HT. Male C57bl/6 mice were sensitised with saline or HDM (no Alum) and subsequently challenged with saline or HDM. Anaesthetised mice were instrumented and airway responsiveness to inhaled 5-HT was assessed by changes in resistance. Data (n = 6) expressed as mean resistance ± S.E.M. (DOC 150 kb)

The requirement for Alum during sensitisation in the allergic inflammatory response to OVA challenge. Male C57bl/6 mice were sensitised with saline or OVA made up in saline or Alum as indicated below the figure. Mice were subsequently challenged with saline (white bars) or OVA (grey bars). 3 days after the last challenge lungs were lavaged and BAL fluid eosinophil (A), neutrophil (B), lymphocyte (C) and total white blood cell (D) numbers were determined. Data expressed as mean cell number (103/ml) ± S.E.M. (n = 7–9). *p

The requirement for Alum during sensitisation in the allergic inflammatory response to OVA challenge. Male C57bl/6 mice were sensitised with saline or OVA made up in saline or Alum as indicated below the figure. Mice were subsequently challenged with saline (white bars) or OVA (grey bars). 3 days after the last challenge lungs were lavaged and BAL fluid eosinophil (A), neutrophil (B), lymphocyte (C) and total white blood cell (D) numbers were determined. Data expressed as mean cell number (103/ml) ± S.E.M. (n = 7–9). *p

The effect of intranasal HDM challenge in topically sensitised mice. Male C57bl/6 mice were sensitised i.n. or i.p. as indicated below the figures with saline or HDM. Mice were subsequently challenged with saline or HDM. 3 days after the last challenge lungs were lavaged and BALF eosinophil (A), neutrophil (B) and lymphocyte (C) numbers were determined. Data (n = 8–12) expressed as mean cell number (103/ml) ± S.E.M. *p

Ig levels in response to sensitisation with HDM via different routes. Male C57bl/6 mice were sensitised with vehicle or HDM (5 μg/kg HDM) either i.n. or i.p. (with or without Alum). Levels of HDM-specific IgE (A), total IgG1 (B) and HDM-specific IgG1 (C) in plasma were measured by ELISA. Data (n = 6) expressed as means ± S.E.M. *p

Investigation of mechanism driving acute inflammatory response to HDM challenge. Naïve male C57bl/6, TLR2, TLR4, Dectin-1 and Dectin-2 KO mice were anaesthetised (4 % isoflurane in oxygen for 3 min) and challenged with i.t. vehicle (saline) or HDM (25 μg/mice in 50 μl saline). 48 h after challenge the lungs were lavaged, lung weight (A) determined, BALF (B) and lung tissue (C) total white blood cell numbers (D) were determined. Data (n = 7–8) expressed as means ± S.E.M. *p

Investigation of mechanism driving acute inflammatory response to HDM challenge. Naïve male C57bl/6, TLR2, TLR4, Dectin-1 and Dectin-2 KO mice were anaesthetised (4 % isoflurane in oxygen for 3 min) and challenged with i.t. vehicle (saline) or HDM (25 μg/mice in 50 μl saline). 48 h after challenge the lungs were lavaged, lung weight (A) determined, BALF (B) and lung tissue (C) total white blood cell numbers (D) were determined. Data (n = 7–8) expressed as means ± S.E.M. *p

The effect of chronic HDM exposure on airway pathology. Male WT C57bl/6 mice were sensitised with saline or HDM and subsequently challenged with HDM 3 times a weeks for 5 weeks. Mice were culled 3 days after final HDM challenge and the lungs collected and assessed for inflammatory status: A) example H&E staining from the control group; B) example H&E staining from the allergic HDM challenged group; C) inflammatory cell burden score; D) mucus production (PAS staining) score; E) collagen deposition (Sirius Red staining) and F) airway smooth muscle area (alpha acting staining). Data (n = 6) expressed as mean ± S.E.M. #p

The effect of chronic HDM exposure on airway pathology. Male WT C57bl/6 mice were sensitised with saline or HDM and subsequently challenged with HDM 3 times a weeks for 5 weeks. Mice were culled 3 days after final HDM challenge and the lungs collected and assessed for inflammatory status: A) example H&E staining from the control group; B) example H&E staining from the allergic HDM challenged group; C) inflammatory cell burden score; D) mucus production (PAS staining) score; E) collagen deposition (Sirius Red staining) and F) airway smooth muscle area (alpha acting staining). Data (n = 6) expressed as mean ± S.E.M. #p