Circulating mitochondrial DNA and Toll-like receptor 9 are associated with vascular dysfunction in spontaneously hypertensive rats

Abstract

Aims: Immune system activation is a common feature of hypertension pathogenesis. However, the mechanisms that initiate this activation are not well understood. Innate immune system recognition and response to danger are becoming apparent in many cardiovascular diseases. Danger signals can arise from not only pathogens, but also damage-associated molecular patterns (DAMPs). Our first hypothesis was that the DAMP, mitochondrial DNA (mtDNA), which is recognized by Toll-like receptor 9 (TLR9), is elevated in the circulation of spontaneously hypertensive rats (SHR), and that the deoxyribonuclease enzymes responsible for its degradation have decreased activity in SHR. Based on these novel SHR phenotypes, we further hypothesized that (i) treatment of SHR with an inhibitory oligodinucleotide for TLR9 (ODN2088) would lower blood pressure and that (ii) treatment of normotensive rats with a TLR9-specific CpG oligonucleotide (ODN2395) would cause endothelial dysfunction and increase blood pressure.

Methods and results: We observed that SHR have elevated circulating mtDNA and diminished deoxyribonuclease I and II activity. Additionally, treatment of SHR with ODN2088 lowered systolic blood pressure. On the other hand, treatment of normotensive rats with ODN2395 increased systolic blood pressure and rendered their arteries less sensitive to acetylcholine-induced relaxation and more sensitive to norepinephrine-induced contraction. This dysfunctional vasoreactivity was due to increased cyclooxygenase and p38 mitogen-activated protein kinase activation, increased reactive oxygen species generation, and reduced nitric oxide bioavailability.

Conclusion: Circulating mtDNA and impaired deoxyribonuclease activity may lead to the activation of the innate immune system, via TLR9, and contribute to elevated arterial pressure and vascular dysfunction in SHR.

Keywords: Hypertension; Innate immunity; Mitochondrial DNA; Toll-like receptor 9; Vascular dysfunction.

Figure 1

Circulating mitochondrial DNA (mtDNA) is elevated in male SHR, and this increased expression is pressure dependent. (A) Quantification of circulating mtDNA genes, cytochrome B (Cyt B), and NADH dehydrogenase subunit 6 (ND6), as measured by RT–PCR from plasma of WKY, SHR, and SHR treated with hydrochlorothiazide and reserpine (HCTZ/Res). 1/Ct denotes the reciprocal of the count where the sequence is detected; a direct function of gene concentration. Bacterial 16S rRNA showed no bacterial contamination of any specimen. (B) Representative blots from endpoint PCR. n = 3–8. One-way ANOVA: *P < 0.05 vs. WKY.

Figure 2

SHR has impaired mitochondrial viability and expression of early apoptosis marker JC-1 in kidney and bone marrow cells. Left, representative histograms for mitochondrial membrane potential (ψm) marker JC-1 monomer in (A) kidney and (B) bone marrow cells of WKY and SHR. Right, densitometric analysis. n = 5. Student's t-test: *P < 0.05 vs. WKY.

Figure 3

SHR has impaired nucleic acid clearance capacity, and expression of autophagic machinery is diminished. Activities of (A) deoxyribonuclease (DNase) I in serum and DNase II in mesenteric resistance arteries and left ventricle from WKY and SHR. Left, representative images of agarose gels. Right, densitometric analysis. Aortic expression of representative autophagy proteins normalized to β-actin: (B) DNase II, (C) Beclin-1, (D) ATG5, and (E) LC3-I and -II. Above, representative images of immunoblots. Below, densitometric analysis. n = 3–6. Student's t-test: *P < 0.05 vs. WKY.

Figure 4

Acute incubation of isolated MRA with TLR9 agonist ODN2395 activates TLR9 signalling. Incubation of naïve Sprague Dawley MRA with TLR9 agonist ODN2395 (2 µmol/L) for 15 or 30 min did not change protein expression of (A) TLR9. However, ODN2395 altered protein expressions of (B) canonical-inflammatory signalling (increased MyD88 and TRAF6) and (C) non-canonical stress tolerance signalling (increased SERCA2 and phosphorylated AMPKα^Thr172), relative to the unstimulated condition. Black bars: unstimulated, dark grey bars: 15 min stimulation, light grey bars: 30 min stimulation, and white bars: 30 min stimulation in the presence of TLR9 antagonist (ODN2088). Below, densitometric analysis. Above, representative images of immunoblots. n = 3–4. One-way ANOVA: *P < 0.05 vs. unstimulated.

Figure 5

TLR9 antagonist ODN2088 lowers SBP in SHR, and TLR9 agonist ODN2395 increases SBP in previously normotensive rats. Systolic blood pressure responses (A) across ODN2088 treatment in WKY and SHR (arrows depict when ODN2088 or Veh were administered) and (B) post-ODN2395 treatment. n = 4–9. Repeated-measures ANOVA (with Bonferroni correction): *P < 0.05 vs. SHR-Veh; one-way ANOVA: *P < 0.05 vs. Veh.

Figure 6

TLR9 agonist ODN2395 impairs endothelium-dependent relaxation in MRA and renders MRA more sensitive to NE via decreased nitric oxide and increased reactive oxygen species. Concentration-response curves for (A) acetylcholine (ACh), (B) sodium nitroprusside (SNP), and (C) NE in MRA from Veh- and ODN2395-treated rats. Concentration-response curves for NE, with or without L-NNA (10⁻⁴ mol/L), in MRA from (D) Veh- and (E) ODN2395-treated rats. (F) Area under the curve (AUC) analysis for NE concentration-response curves, with or without L-NNA. Concentration-response curves for NE, with or without tempol (10⁻³ mol/L), in MRA from (G) Veh- and (H) ODN2395-treated rats. (I) AUC analysis for NE concentration-response curves, with or without tempol. (J) Dihydroethidium (DHE) fluorescent staining (×20 magnification) in MRA from Veh- and ODN2395-treated rats, with or without L-NNA (10⁻⁴ mol/L). Left, representative images of fluorescent staining. Right, densitometric analysis. (K) MRA expression of phosphorylated eNOS^Ser1177 normalized for total eNOS. Left, representative images of immunoblot. Right, densitometric analysis. n = 6/6 (independent rats/MRA segments). LogEC₅₀ or Student's t-test: *P < 0.05 vs. Veh; LogEC₅₀: ^#P < 0.05 vs. ODN2395.

Figure 7

Cyclooxygenase (COX) and p38 MAPK inhibition normalizes ODN2395-induced sensitivity to NE in MRA. Concentration-response curves for NE, with or without indomethacin (10⁻⁵ mol/L), in MRA from (A) Veh- and (B) ODN2395-treated rats. (C) Area under the curve (AUC) analysis for NE concentration-response curves, with or without indomethacin. MRA expression of (D) COX 2 and (E) COX 1 normalized to β-actin. Above, representative images of immunoblots. Below, densitometric analysis. Concentration-response curves for NE in MRA from (F) Veh- and (G) ODN2395-treated rats with or without SB203580 (10⁻⁵ mol/L). (H) Phosphorylated p38^{Thr180/Tyr182} MAPK normalized for total p38 MAPK. Above, representative images of immunoblot. Below, densitometric analysis. n = 6/6 (independent rats/MRA segments). Student's t-test: *P < 0.05 vs. Veh; LogEC₅₀: ^#P < 0.05 vs. ODN2395.