Increased arachidonic acid-induced thromboxane generation impairs skeletal muscle arteriolar dilation with genetic dyslipidemia

Abstract

Objective: The aim of this study was to determine if arachidonic acid (AA)-induced skeletal muscle arteriolar dilation is altered with hypercholesterolemia in ApoE and low-density lipoprotein receptor (LDLR) gene deletion mice fed a normal diet. This study also determined contributors to altered AA-induced dilation between dyslipidemic mice and controls, C57/Bl/6J (C57).

Methods: Gracilis muscle arterioles were isolated, with mechanical responses assessed following a challenge with AA under control conditions and after elements of AA metabolism pathways were inhibited. Conduit arteries from each strain were used to assess AA-induced production of PGI(2) and TxA(2).

Results: Arterioles from ApoE and LDLR exhibited a blunted dilation to AA versus C57. While responses were cyclo-oxygenase-dependent in all strains, inhibition of thromboxane synthase or blockade of PGH(2)/TxA(2) receptors improved dilation in ApoE and LDLR only. AA-induced generation of PGI(2) was comparable across strains, although TxA(2) generation was increased in ApoE and LDLR. Arteriolar reactivity to PGI(2) and TxA(2) was comparable across strains. Treatment with TEMPOL improved dilation and reduced TxA(2) production with AA in ApoE and LDLR.

Conclusions: These results suggest that AA-induced arteriolar dilation is constrained in ApoE and LDLR via an increased production of TxA(2). While partially due to elevated oxidant stress, additional mechanisms contribute that are independent of acute alterations in oxidant tone.

Figure 1

Data describing the dilator reactivity of isolated skeletal muscle resistance arterioles of C57, ApoE and LDLR mice in response to increasing concentrations of arachidonic acid. Data, presented as mean±SEM, are shown for arterioles under control conditions and following removal of the vascular endothelium using air bolus perfusion (please see text for details). n=6 animals for each strain; * p<0.05 vs. C57; † p<0.05 vs. control within that strain.

Figure 2

Data describing the dilator responses of isolated skeletal muscle resistance arterioles of C57 (Panel A), ApoE (Panel B) and LDLR (Panel C) mice in response to increasing concentrations of arachidonic acid. Data, presented as mean±SEM, are shown for arterioles under control conditions, and following pharmacological inhibition of cyclooxygenases with indomethacin, lipoxygenases with NDGA or combined inhibition of both enzymatic pathways (please see text for details). n=5–10 animals for each group; * p<0.05 vs. control conditions, † p<0.05 vs. no response.

Figure 2

Data describing the dilator responses of isolated skeletal muscle resistance arterioles of C57 (Panel A), ApoE (Panel B) and LDLR (Panel C) mice in response to increasing concentrations of arachidonic acid. Data, presented as mean±SEM, are shown for arterioles under control conditions, and following pharmacological inhibition of cyclooxygenases with indomethacin, lipoxygenases with NDGA or combined inhibition of both enzymatic pathways (please see text for details). n=5–10 animals for each group; * p<0.05 vs. control conditions, † p<0.05 vs. no response.

Figure 2

Data describing the dilator responses of isolated skeletal muscle resistance arterioles of C57 (Panel A), ApoE (Panel B) and LDLR (Panel C) mice in response to increasing concentrations of arachidonic acid. Data, presented as mean±SEM, are shown for arterioles under control conditions, and following pharmacological inhibition of cyclooxygenases with indomethacin, lipoxygenases with NDGA or combined inhibition of both enzymatic pathways (please see text for details). n=5–10 animals for each group; * p<0.05 vs. control conditions, † p<0.05 vs. no response.

Figure 3

Data describing the dilator responses of isolated skeletal muscle resistance arterioles of C57 (Panel A), ApoE (Panel B) and LDLR (Panel C) mice in response to increasing concentrations of arachidonic acid. Data, presented as mean±SEM, are shown for arterioles under control conditions, and following pharmacological inhibition of PGH₂/TxA₂ receptors with SQ-29548 and thromboxane synthase with CHI (please see text for details). n=6–7 animals for each group; * p<0.05 vs. control conditions.

Figure 3

Data describing the dilator responses of isolated skeletal muscle resistance arterioles of C57 (Panel A), ApoE (Panel B) and LDLR (Panel C) mice in response to increasing concentrations of arachidonic acid. Data, presented as mean±SEM, are shown for arterioles under control conditions, and following pharmacological inhibition of PGH₂/TxA₂ receptors with SQ-29548 and thromboxane synthase with CHI (please see text for details). n=6–7 animals for each group; * p<0.05 vs. control conditions.

Figure 3

Data describing the dilator responses of isolated skeletal muscle resistance arterioles of C57 (Panel A), ApoE (Panel B) and LDLR (Panel C) mice in response to increasing concentrations of arachidonic acid. Data, presented as mean±SEM, are shown for arterioles under control conditions, and following pharmacological inhibition of PGH₂/TxA₂ receptors with SQ-29548 and thromboxane synthase with CHI (please see text for details). n=6–7 animals for each group; * p<0.05 vs. control conditions.

Figure 4

Data describing the arterial production of prostacyclin (as 6-keto-PGF_1α; Panel A) or thromboxane A₂ (as 11-dehydro TxB₂; Panel B) from C57, ApoE and LDLR in response to 10⁻⁶ M arachidonic acid. Data, presented as mean±SEM, are shown under control conditions, and following pharmacological inhibition of cyclooxygenase with indomethacin or thromboxane synthase (with CHI), as appropriate. n=8 animals for each group, with each n representing pooled arteries from an individual mouse; please see text for details. *p<0.05 vs. respective control; † p<0.05 vs. C57 under that condition; ‡ vs. ApoE under that condition.

Figure 4

Data describing the arterial production of prostacyclin (as 6-keto-PGF_1α; Panel A) or thromboxane A₂ (as 11-dehydro TxB₂; Panel B) from C57, ApoE and LDLR in response to 10⁻⁶ M arachidonic acid. Data, presented as mean±SEM, are shown under control conditions, and following pharmacological inhibition of cyclooxygenase with indomethacin or thromboxane synthase (with CHI), as appropriate. n=8 animals for each group, with each n representing pooled arteries from an individual mouse; please see text for details. *p<0.05 vs. respective control; † p<0.05 vs. C57 under that condition; ‡ vs. ApoE under that condition.

Figure 5

Data (mean±SEM) describing the reactivity of isolated skeletal muscle resistance arterioles of C57, ApoE and LDLR mice in response to increasing concentrations of prostacyclin (Panel A) or carbocyclic thromboxane A₂ (Panel B). n=6 animals for each group, no significant differences were identified in the vascular reactivity in response to increasing concentrations of prostacyclin or thromboxane A₂.

Figure 5

Data (mean±SEM) describing the reactivity of isolated skeletal muscle resistance arterioles of C57, ApoE and LDLR mice in response to increasing concentrations of prostacyclin (Panel A) or carbocyclic thromboxane A₂ (Panel B). n=6 animals for each group, no significant differences were identified in the vascular reactivity in response to increasing concentrations of prostacyclin or thromboxane A₂.

Figure 6

Data, presented as mean±SEM, describing the dilator responses of isolated skeletal muscle resistance arterioles of C57 (Panel A), ApoE (Panel B) and LDLR (Panel C) mice in response to increasing concentrations of arachidonic acid. Data are shown for arterioles under control conditions, following treatment of vessels with the antioxidant TEMPOL, following pharmacological inhibition of thromboxane synthase with CHI, and following treatment with both TEMPOL and CHI. n=8–10 animals for each group; * p<0.05 vs. control conditions; † p<0.05 vs. treatment with TEMPOL alone.

Figure 6

Data, presented as mean±SEM, describing the dilator responses of isolated skeletal muscle resistance arterioles of C57 (Panel A), ApoE (Panel B) and LDLR (Panel C) mice in response to increasing concentrations of arachidonic acid. Data are shown for arterioles under control conditions, following treatment of vessels with the antioxidant TEMPOL, following pharmacological inhibition of thromboxane synthase with CHI, and following treatment with both TEMPOL and CHI. n=8–10 animals for each group; * p<0.05 vs. control conditions; † p<0.05 vs. treatment with TEMPOL alone.

Figure 6

Data, presented as mean±SEM, describing the dilator responses of isolated skeletal muscle resistance arterioles of C57 (Panel A), ApoE (Panel B) and LDLR (Panel C) mice in response to increasing concentrations of arachidonic acid. Data are shown for arterioles under control conditions, following treatment of vessels with the antioxidant TEMPOL, following pharmacological inhibition of thromboxane synthase with CHI, and following treatment with both TEMPOL and CHI. n=8–10 animals for each group; * p<0.05 vs. control conditions; † p<0.05 vs. treatment with TEMPOL alone.

Figure 7

Data describing the arterial production of thromboxane A₂ (as 11-dehydro TxB₂; Panel B) from C57, ApoE and LDLR in response to 10⁻⁶ M arachidonic acid. Data, presented as mean±SEM, are shown under control conditions, and following treatment of pooled arteries with the antioxidant TEMPOL (10⁻⁴ M). n=6 animals for each group, with each n representing pooled arteries from an individual mouse; please see text for details. * p<0.05 vs. within-strain/no arachidonic acid; † p<0.05 vs. within-strain/with arachidonic acid.