Role of macrophage PPARγ in experimental hypertension

Abstract

Targeted disruption of the Alox15 gene makes mice resistant to angiotensin II-, DOCA/salt-, and N(ω)-nitro-L-arginine methyl ester (L-NAME)-induced experimental hypertension. Macrophages, a primary source of Alox15, are facilitating this resistance, but the underlying mechanism is not known. Because Alox15 metabolites are peroxisome proliferator-activated receptor (PPAR)γ agonists, we hypothesized that activation of macrophage PPARγ is the key step in Alox15 mediation of hypertension. Thioglycollate, used for macrophage elicitation, selectively upregulated PPARγ and its target gene CD36 in peritoneal macrophages of both wild-type (WT) and Alox15(-/-) mice. Moreover, thioglycollate-injected Alox15(-/-) mice became hypertensive upon L-NAME treatment. A similar hypertensive effect was observed with adoptive transfer of thioglycollate-elicited Alox15(-/-) macrophages into Alox15(-/-) recipient mice. The role of PPARγ was further specified by using the selective PPARγ antagonist GW9662. WT mice treated with 50 μg/kg daily dose of GW9662 for 12 days became resistant to L-NAME-induced hypertension. The PPARγ antagonist treatment also prevented L-NAME-induced hypertension in thioglycollate-injected Alox15(-/-) mice, indicating a PPARγ-mediated effect in macrophage elicitation and the resultant hypertension. These results indicate a regulatory role for macrophage-localized PPARγ in L-NAME-induced experimental hypertension.

Keywords: alox15; lipoxygenase; nitro-l-arginine methyl ester; peritoneal macrophages; peroxisome proliferator-activated receptor γ; thioglycollate.

Fig. 1.

Cell type distribution and Alox15 protein expression in peritoneal lavages. A: shift in the ratios of macrophages and lymphocytes between thioglycollate-elicited (black bars) and nonelicited (gray bars) peritoneal lavages as measured by flow cytometry. B: Western immunoblot analysis of Alox15 protein expression in sorted lymphocyte and macrophages of elicited peritoneal lavage. PM, peritoneal macrophages; T, T lymphocytes; B, B lymphocytes. β-Actin was used as a loading control. Values are means ± SE; n = 3. Representative blots of 3 experiments are shown.

Fig. 2.

Effect of thioglycollate-elicited Alox15^−/− macrophages on N^ω-nitro-l-arginine methyl ester (l-NAME)-induced hypertension in Alox15^−/− mice. Hypertension was induced with l-NAME treatment in wild-type (WT) or Alox15^−/− mice and in Alox15^−/− recipient mice that were injected with 8 × 10⁶ thioglycollate-elicited Alox15^−/− macrophages. Blood pressure was monitored by the tail-cuff method. Each value represents mean ± SE; n = 4 for PM- or vehicle-injected Alox15^−/− mice and n = 8 for all other groups. *P < 0.01 compared with control.

Fig. 3.

Effect of thioglycollate elicitation on peroxisome proliferator-activated receptor (PPAR)γ and CD36 protein expression in peritoneal macrophages and T cells. Macrophages and T cells from control (−TG) or thioglycollate-injected (+TG) WT and Alox15^−/− mice were harvested and subjected to Western immunoblot analysis. β-Actin was used as a loading control. Representative blots of 3 experiments for Alox15^−/− and 4 experiments for WT cells are shown. Relative densitometric values for each Western immunoblot of PPARγ (left) or CD36 (right) are shown. *P < 0.01 compared with nonelicited control; **P < 0.001 compared with nonelicited control.

Fig. 4.

Effect of PPARγ inhibition on development of l-NAME-induced hypertension in WT (A), Alox15^−/− (C), and thioglycollate-injected Alox15^−/− (D) mice. Hypertension was induced with l-NAME after 12 days of daily PPARγ inhibitor (GW9662) injections. Alox15^−/− mice (D) were injected with thioglycollate before the start of the l-NAME treatment. Blood pressure was monitored by the tail-cuff method. Each value represents mean ± SE; n = 4 for TG-injected Alox15^−/− mice and n = 6 for WT or Alox15^−/− mice without TG injection. B: effect of GW9662 treatment on PPARγ activity of PM of WT mice that have been elicited with thioglycollate. Representative Western immunoblots of 3 experiments are shown. β-Actin was used as a loading control. Relative densitometric values for Western immunoblot are shown. *P < 0.005 compared with control; **P < 0.005 compared with vehicle + l-NAME group.

Fig. 5.

Effect of thioglycollate (TG) injection on PPARγ and CD36 protein expression in aorta, PM, and kidney as detected by Western immunoblot analysis of 50 μg/lane total lysate proteins. Top: representative Western immunoblots of PPARγ (left) and CD36 (right) of 3 experiments. Bottom: densitometric values from the blots at top represented as means ± SE of 3 experiments. *P < 0.001 compared with noninjected control. β-Actin was used as a loading control.

Fig. 6.

Schematic overview of the role of macrophage-localized Alox15/PPARγ pathway in the development of l-NAME-induced hypertension. In WT mice (left), Alox15 (12/15-LO) generates endogenous PPARγ agonists 15-hydroxyeicosatetraenoic acids (15-HETE) or 13-hydroxyoctadecadienoic acids (13-HODE) from arachidonic acid (AA) or linoleic acid (LA), respectively, which activate PPARγ, increasing the expression of PPARγ-regulated genes such as CD36 and l-NAME-induced hypertension. Hypertension develops. Genetic disruption of the Alox15 gene (middle) eliminates the synthesis of these PPARγ agonists, which downregulates PPARγ and PPARγ-regulated genes like CD36. Hypertension does not occur with l-NAME. In Alox15^−/− mice (right), externally added PPARγ agonists such as thioglycollate, 15-HETE/13-HODE, or other unknown compounds dramatically upregulate both PPARγ and PPARγ-regulated genes like CD36, restoring the development of l-NAME hypertension. NO, nitric oxide; LO, lipoxygenase.